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Methods for gender determination and selection of avian embryos in unhatched eggs

a technology of avian embryos and gender determination, which is applied in the field of non-invasive methods and transgenic avian animals for gender determination and selection of embryos in unhatched avian eggs, and achieves the effects of high efficiency and selectiveness, and great proficiency

Inactive Publication Date: 2020-05-14
EGGXYT LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is a method that uses transgenic avians to select a desired gender and produce male-free flocks. The method involves breeding pairs of avians that have complementing CRISPR-Cas components that knock out a gene crucial for cell survival and development. The method also includes an initial selection step where a reporter gene, such as a fluorescent protein, is integrated into the genome of the mother or the father. The method is highly efficient and selective, with more than 99% effectiveness in eliminating non-fertilized eggs, eggs with male embryos, or both, as early as 5 days post egg laying.

Problems solved by technology

Each day, billions of male chicks are being terminated via suffocation or grinding since they are not useful for laying eggs or to be bread for meat.
Effective and non-invasive methods for sex identification and subsequent selection during the egg stage, prior to the hatching of the chick are currently not available.

Method used

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  • Methods for gender determination and selection of avian embryos in unhatched eggs
  • Methods for gender determination and selection of avian embryos in unhatched eggs
  • Methods for gender determination and selection of avian embryos in unhatched eggs

Examples

Experimental program
Comparison scheme
Effect test

example 1

Obtaining an Avian Transgenic Cell

[0214]The process includes knocking-in a reporter gene (CMV-RFP) to the designated genome loci, which is described hereinbelow.

Preparation of gRNA Duplex:

[0215]Components: Z-specific gRNA oligo (ChrZgRNA sequence: 5′-GUAAUACAGAGCUAAACCAG-3′; SEQ ID NO: 30); tracrRNA (IDT cat #1072533); and Nuclease-Free Duplex Buffer (IDT cat #11-01-03-01).

[0216]RNA oligos are resuspend in Nuclease-Free Duplex Buffer to a final concentration of 100 μM (Micromolar) (for 10 nM (nanomolar) gRNA use 100 μl of Nuclease free buffer). Z-specific gRNA and tracrRNA are mixed in equimolar concentrations to create a final duplex concentration of 1 μM from each oligo (the duplex can be stored in −20° C. for multiple uses). Duplexes are heated at 95° C. for 5 min, and thereafter are cooled to room temperature (20-25 ° C.).

Preparation of RNP Complex and Transfection:

[0217]Components: HiFi Cas9 Nuclease V3 (IDT cat #1081061); Opti-MEM reduced serum media (Thermo cat #11058021); LI...

example 2

Obtaining a Transgenic Embryo / Organism

[0220]In order to generate transgenic chick, primordial germ cells (PGCs) are extracted, manipulated and retrieved to another embryo for the purpose of germ line chimera generation.

Collection of 18 Hamburger Hamilton (HH) Stage Embryonic Blood for the Purpose of PGC Isolation

[0221]Fertilized eggs are incubated in 39° C. incubator for 72 hrs. Following the 72 hrs incubation, eggs are removed from the incubator, and each egg is punctured using a 19 G needle. Three (3) to 5 ml of albumin is drawn from each egg. A “window” is opened in each egg by carefully removing a part of shell. A heated 5 μl pointed capillary is used to cause controlled bleeding from the embryo's vascular system. Up to 5 μl of embryonic blood is collected and deposited into 48-wells plate (48 W) filled with Avian DMEM media. After 1-2 weeks, red blood cells die and PGCs are visible. Fresh medium is replenished every 3 or 4 day. When PGCs reach confluency in the 48 W, they are t...

example 3

Validation of Genome Editing (GE) Event

Validation of Genome Cleavage Using T7EI Enzyme Assay

[0226]Components: GENEART™ Genomic Cleavage Detection Kit (Thermo cat#A24372); gRNA: Cas9 transfected DF1 cells (an ATCC cell line #CRL12203); and detection primers for cleavage with ChrZgRNA: Forward: 5′-GCGGAGGGAGATGAAACA-3′ (SEQ ID NO: 31), and Reverse: 5′-AAGGGATCACACAGCTCAAG-3′ (SEQ ID NO: 32).

[0227]Hundred (100) μl of cell lysis buffer (GeneArt Kit) are mixed with 4 μl of protein degrader (GeneArt Kit), in a microcentrifuge tube. Culture media is removed from transfected cells, which are then added with 100 μl of the cell lysis mix. The cells are then incubated at room temperature for 5 min. Lysed cells are then collected and transfer into a PCR (0.2 μl) tube. The lysed cells are placed in a thermo-cycler and processed according to the following program: 68° C. for 15 min, 95° C. for 10 min, and hold on 4° C. Thereafter, an amplification reaction is set according to the following table:...

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Abstract

The present invention, in some embodiments thereof, is directed to a method for selecting a gender of an avian fertilized unhatched egg obtained from the crossing of a transgenic male avian subject and a transgenic female avian subject. Further provided is a kit for preparing a transgenic avian subject.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation-in-part of U.S. patent application Ser. No. 15 / 996,045, filed Jun. 1, 2018, titled “METHODS FOR GENDER DETERMINATION OF AVIAN EMBRYOS IN UNHATCHED EGGS AND MEANS THEREOF”, which is a continuation-in-part of International Patent Application No. PCT / IL2016 / 051291, filed Dec. 1, 2016, titled “METHODS FOR GENDER DETERMINATION OF AVIAN EMBRYOS IN UNHATCHED EGGS AND MEANS THEREOF”, which claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 262,409, filed Dec. 3, 2015, titled “A METHOD FOR GENDER SELECTION OF UNHATCHED EGGS BY BIOLUMINESCENCE”.[0002]This application is also a continuation-in-part of U.S. patent application Ser. No. 16 / 616,858, filed Nov. 25, 2019, titled “METHODS FOR GENDER DETERMINATION OF AVIAN EMBRYOS IN UNHATCHED EGGS AND MEANS THEREOF”, which is a national phase of International Patent Application No. PCT / IL2018 / 050573, filed May 24, 2018, titled “METHODS FOR GENDER...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12Q1/6879C12Q1/6897A01K67/027
CPCC12Q1/6879A01K2217/05A01K2227/30A01K67/0275C12N15/8509C12Q1/6897C12Q2600/124A01K2217/07C12N15/873
Inventor OFFEN, DANIELELRAM, YEHUDA
Owner EGGXYT LTD
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